The accurate segregation of chromosomes during mitosis is critical to eucaryotic cell division. Immunological and genetic approaches will be combined to identify chromosome segregation components in the model eucaryote; Saccharomyces cerevisiae. Human autoantibodies from scleroderma patients react with conserved determinants of the mammalian spindle pole (centrosome). These have been used to identify two related antigens in yeast. The subcellular localization of these and other potential antigens within a yeast cell will be determined by indirect immunofluorescence. Genes encoding these proteins will be cloned by immunoscreening a Lambdagt11 yeast genomic DNA library. The genes will be characterized and sequenced and antibodies raised against the gene products. Mutations will be constructed in these genes (in vitro or in E. coli), which will then be substituted for the genomic copies in vivo. This procedure will utilize a novel method for rapidly and crudely mapping antigenic coding regions, and creating insertion mutations using transposition mutagenesis. The phenotypes of these mutations on cell viability and growth, chromosome segregation, zygote formation, and meiosis will be examined. Conditional mutations will be sought that allow the isolation of extragenic second site suppressors. In addition to the immunological approach, a genetic screen will be performed to identify mutations in genes involved in chromosome segregation. This screen uses a colony color assay to identify mutants that exhibit a high frequency of chromosome nondisjunction. A restricted subset of mutants, with phenotypes expected of chromosome segregation mutants will be studied extensively. The combined immunological and genetic approaches will identify components involved in eucaryotic chromosome segregation. Biochemical and genetic analysis of these components in yeast provide a unique opportunity to examine the molecular events that occur during mitosis, including the mechanism of action of individual proteins, the proteins with which they interact, and how the assembly and disassembly of the mitotic apparatus is controlled in the eucaryotic cell cycle. Scleroderma is an autoimmune disease of unknown etiology and unknown pathogenicity. This proposal will identify, for the first time, molecules recognized by these spindle pole scleroderma sera. This information and these approaches will be useful for understanding etiology, pathogenicity and diagnosis of autoimmune disease.